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Plant biology – Retrospect and prospect
Arthur W. Galston
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, 06520-8103, USA
Over the last century, plant biology has made remarkable progress, reflected in its greatly improved
journals and textbooks. This article summarizes
some historically important developments in the
study of plant photoreceptors, especially the redabsorbing phytochromes and blue-absorbing flavoproteins, in phytohormones, in cell and tissue culture
and in circadian rhythms. Attention is drawn to the
neglect of polyamines, especially in relation to stress.
Some effects of altered patterns of research support
are noted, and a few future research trends are suggested as probable; these include increased use of
molecular genetic techniques and increased attention
to agricultural, ecological and ethical problems.
T AKING the pulse of experimental plant biology at the
turn of the millennium and noting some of the changes
over recent time is a daunting challenge, undertaken
here with trepidation. Since one obviously cannot cover
all subjects, what follows must represent a subjective
and highly personal selection. It is limited to the 20th
century, into which I was born almost eighty years ago,
at about the time of the formation of the American Society of Plant Physiologists. My birth also virtually coincided with Garner and Allard’s landmark article on
photoperiodism1, and occurred just a few years before
Frits Went’s discovery of auxin2 ushered in the era of
phytohormones. Since my own research involved these
two areas, I will dwell on some historical developments
in both.
How things were back then
In the year l936, I entered Cornell University as a
freshman and began my first studies in Botany. The
Cornell Botany Department was at that time one of the
best in the world, especially strong in maize genetics
and cytology, thanks to the presence of such luminaries
as R. A. Emerson and the future Nobelists G. W. Beadle
and Barbara McClintock. The Department also boasted
great strength in anatomy and comparative morphology
(A. J. Eames and L. H. MacDaniels), and in physiology
(L. Knudson, O. F. Curtis and D. G. Clark). Yet as I
look back on my student notes of those days, I am astounded at how old-fashioned many of those thoughts
e-mail: [email protected]
CURRENT SCIENCE, VOL. 80, NO. 2, 25 JANUARY 2001
and procedures seem today, and as a consequence, what
an inadequate preparation such training would be for
the biology of today. The training of modern plant biologists is both broader in scope and more rigorous than
I ever experienced, auguring well for an enhanced quality of work and a higher status for our field in the future. One possible modern problem is that the intensive
training required for proficiency in the molecular areas
tends to produce many narrow specialists lacking the
historical background that yields a broad perspective of
our science.
In the mid-1930s, we botanists paid scant attention to
metabolism, or in fact to any aspect of plant biochemistry, and the biochemistry course I took in another department scarcely mentioned plants. The study of plant
hormones, which had arisen out of the study of tropisms
in grass coleoptiles, was in its infancy, with only impure preparations of chemically uncharacterized ‘auxin’
available for experimentation and speculation. During
my four undergraduate years at Cornell, I took all the
plant physiology courses available, including elementary and advanced plant physiology and laboratory
techniques; yet during all this time, I never experienced
a single lecture or a laboratory exercise dealing with
plant hormones. The most frequent lecture subjects revolved around the uptake, transport and transpiration of
water, xylem vs phloem transport and mineral absorption and nutrition. Mirroring and perhaps directing this
selection of topics was the textbook in general use at
that time, Plant Physiology by Miller3, a ponderous and
rather formidable compendium of a large volume of
observations, data and theories. That situation contrasts
sharply with a similarly entitled modern text by Taiz
and Zeiger4, in which selected and restricted data are
introduced only if relevant to clearly delineated theories
or phenomena. Similarly, considered against the standards of our modern research journals, the plant biological journals at the start of the century seem
primitive and unpolished.
It was not until I began graduate study at the University of Illinois in 1940 that I received my first lectures
on such subjects as plant hormones, photoperiodism and
vernalization from H. J. Fuller, and some laboratory
training in biochemistry under H. E. Carter of the eminent University of Illinois Chemistry Department. As I
took a joint major in botany and biochemistry, my dissertation committee consisted of representatives of both
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groups. As a result, my defence of thesis on the physiology of flowering was almost ridiculously easy, since
the botanists were convinced that my biochemistry was
quite sophisticated and the biochemists, essentially innocent of training in botany, thought the same about my
botanical expertise. One prominent biochemist on the
committee, William C. Rose, confessed that he had not
known whether the word ‘photoperiodism’ referred to
effects of light periodicity or to light effects on iodine
metabolism (photo-per-iodism)!
Since biochemical training was rarely offered to the
plant physiologists of that day, there existed considerable tension between plant physiologists and plant biochemists, tension of the sort that originally separated
classical botany from plant physiology and that now
partially separates plant molecular biology from the rest
of the field. In each instance, I am heartened to note, the
original mutual suspicion between specialties led later
to a strengthening of the entire field by incorporation of
the newer, more radical discipline into the mainstream
of the subject. Perhaps it is not too much to hope that
the gap that presently separates plant molecular biology
from some of the rest of the field will be similarly
closed with time.
Although not noted as an experimentalist, Harry
Fuller was an eloquent and inspirational teacher, who
opened my eyes to the fields of flowering physiology as
related to hormones, circadian rhythms and in general,
the influence of light on plant behaviour. Although frequently absent from the campus during the hectic years
of World War II, when he was exploring South America
for stands of Hevea brasiliensis that might ameliorate
the serious shortage of rubber resulting from the Japanese conquest of the Malaysian peninsula, he conscientiously and devotedly directed my thesis on the
physiology of flowering in soybeans. (Incidentally in
Arthur W. Galston
Arthur W. Galston is among the most distinguished and respected plant biologists of our times, who has become a
legend in his own life time. He has been an outstanding teacher who held generations of students spell-bound
through his most wonderful, lively and thought-provoking lectures. His landmark books have changed the course of
many careers. Written in simple and engaging style, these books have been
translated in many languages and students from all over the world, and over
the years have thronged to his laboratory to carve out a career in plant biology. However, books comprise a small part of his total contribution – his research programmes have had a profound influence on the growth of plant
biology.
Galston had humble beginnings. The lives of his parents were catapulted as they had to escape the anti-semitic excesses of Czarist Russia
and flee to USA at the turn of the last century. He was born in 1920 in
Brooklyn, New York where he had his schooling. In his own words: ‘I aspired to study medicine but that entailed expenses that were far beyond
the capacity of my family. So I chose veterinary medicine instead, since
there was a tuition-free college for New York State residents on the campus of Cornell University. However, this career was de-railed by a charismatic professor, Loren C. Petry, who taught me elementary botany.’
Galston majored in plant physiology, receiving his BS in 1940. Readers
would be interested to know that utilizing his musical talents the young
Galston earned his way through college, playing the saxophone! He was
awarded the Ph D degree in 1943 for his seminal work on the physiology
of flowering. Again, in his own words: ‘Expecting to enter military service
immediately after completion of my thesis, I was surprised to receive an
offer of a scientific job, and thus yielding a deferment of service. I went to work for James Bonner at CalTech
on the Emergency Rubber Project, designed to produce rubber from the Mexican shrub, guayule (Parthenium
argentatum Gray). This respite lasted not quite one year, after which I entered the navy as an enlisted man;
later on I became a commissioned officer and saw my major duty on a Military Government team on Okinawa.
I was released from service after two years, and returned to New York City.’ But only briefly. After one year’s
instructorship at Yale, Galston again went back to CalTech and this time for nine years. These according to
him were the best years of his life as he had an opportunity to interact with the intellectual giants of our time,
Max Delbruck, Richard Feynman, Linus Pauling and also outstanding biologists – George Beadle, James Bonner, Frits Went. The next 45 years of his very productive life were spent at Yale. His latest interest is bioethics,
the beneficiaries of his interest are Yale undergraduates. Because of the great relevance of bioethics today, it
is hoped that Galston’s wisdom would spread far and wide.
In the accompanying article Galston traces the growth of plant biology and this he does in the first person.
(SCM and SKS)
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1940, when I began my studies, this crop, now our major agricultural export, was planted only experimentally,
and on only a few Illinois farms!). Fuller had recently
returned from a sabbatical spent at CalTech with Frits
Went (my future colleague), and introduced me to the
intricacies of the Avena bioassay, then the standard
method for estimating auxin quantitatively. I performed
hundreds of Avena test bioassays in the course of my
thesis research. The interest in such subjects generated
by this early work has remained with me all of my life,
manifesting itself in much of my research on photoreactions in plants and on hormone physiology.
Support of scientific research: A changing scene
When I began my graduate work at the University of
Illinois in September l940, the country was still trying
to pull itself out of a prolonged economic depression
which had started a little over a decade earlier. Unemployment was still a problem, and graduate assistantships and fellowships were greatly prized and highly
competitive. Science was usually done on a ‘shoestring’
budget, with extremely simple equipment. World War II
changed all that in the United States; suddenly there
was no significant unemployment, support of young
scientists became more common and organized governmental support of research science at universities became regularized, at first through military agencies like
the Office of Naval Research, later through the National
Science Foundation, the National Institutes of Health,
the Department of Energy, the Department of Agriculture, the National Aeronautics and Space Agency, and
numerous smaller sources of funds. At the same time,
private foundations like Rockefeller and Ford added
their tremendous assets to the support of scientific research.
From about 1950 to 1990, financial support was
available to almost all able investigators in the United
States, leading to a veritable flood of scientific findings.
Soon, agencies in Europe, Japan and elsewhere enlarged
their activities, providing a similar stimulus in their own
countries: the medical and agricultural research groups
(MRC and ARC) in the UK, the Deutsche Forschungsgemeinschaft and the Max Planck Institute in Germany,
the CNRS in France, RIKEN in Japan and a host of
similar organizations in other countries arose to support
scientific research, which was perceived as an investment that ‘paid off’ in the future. Private industry, also
sensing the advantages of sponsored research, added to
its own in-house efforts, and also subsidized relevant
activities in academe. For the academic researcher, this
new money was a boon, permitting the planning and
execution of many scientific projects which would otherwise have remained in limbo.
CURRENT SCIENCE, VOL. 80, NO. 2, 25 JANUARY 2001
Like almost all desirable innovations, this change in
the support of science has also had some unfortunate
consequences. One serious side-effect has been that
many able senior investigators have been effectively
removed from direct participation in active research,
spending their time mainly in the preparation of successful grant applications, the hiring of effective postdoctoral fellows, the recruiting of able graduate students
and the writing of papers worthy of publication in respected journals. After a decade or so of such isolation
from the laboratory, some investigators may well have
run out of useful ideas or lost the desire or ability to do
research.
Important research trends
In my view, there has been no more important or unique
series of investigations in 20th century plant physiology
than those leading to the discovery of phytochrome and
subsequent exposition of its physiological significance.
I shall thus start with this subject, then move on to the
related subjects of phototropism and blue light effects,
then to tissue culture, plant hormones and circadian
rhythms.
Photoreceptor pigments
Although the receptor pigments for photosynthesis have
long been known as chlorophylls, carotenoids and other
pigments capable of transferring their excitation energy
to the photochemical centres of the chloroplast, knowledge of the photoreceptors for processes directly linked
to growth and development have remained mysterious
until comparatively recently. However, spectacular successes have now been achieved in identifying the pigments responsible for photon absorption for mainly redlight-sensitive (photoperiodism, deetiolation, etc.) and
blue-light-sensitive (phototropism, photoreactivation)
reactions in plants. In fact, the discoveries of phytochrome and of photoreactive flavoproteins have radically transformed our understanding of many different
processes studied in modern plant physiology.
Phytochrome: Phytochrome was discovered in 1959
thanks to the genius of a physical chemist, Sterling B.
Hendricks, working in conjunction with several botanists, including Harry A. Borthwick, at the United
States Department of Agriculture (USDA) Station in
Beltsville, Maryland, near Washington DC. Planning to
extend the discoveries of their USDA predecessors
W. W. Garner and H. A. Allard, who had uncovered
major facts about the photoperiodic control of flowering1, and utilizing the finding of Hamner and Bonner
that photoperiodically sensitive plants measure the
length of the dark, rather than of the light period5,
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Hendricks et al.6 constructed a large monochromator,
using parts scavenged from other laboratories and defunct theatres. With this apparatus, they were able to
expose sets of photoperiodically determinate plants to
selected energies and wavelengths in the middle of their
dark period, thus favouring the flowering of long-day
plants and inhibiting the flowering of short-day plants.
By producing dose–response curves for each selected
wavelength, they were able to determine an action spectrum for the interruption of flowering in a short-day
plant (soybean) and the promotion of flowering in a
long-day plant (barley). These two virtually identical
action spectra7, showing major peaks in the red and
smaller peaks in the blue, resembled the absorption
spectrum of a known pigment, allo-phycocyanin, an
open-chain, metal-free tetrapyrrole8. But this pigment,
found readily in the cyanobacteria, could not be shown
to exist in higher plants. It was here that serendipidity
intervened. A previous experiment by Flint and McAlister9 at the nearby Smithsonian Institution Radiation
Laboratory in Washington DC. on the photoactivation
of lettuce-seed germination, had produced a similar action spectrum, peaking at about 660 nm. A surprising
added feature of this action spectrum, discovered because the ‘controls’ had been poised at about 50% germination, was a marked inhibition of germination by
‘far-red’ light, peaking at about 730 nm. Hendricks and
colleagues then showed that far-red given after red annulled the promotive effects of red, and that red given
after far-red exposures would similarly reverse the latter’s inhibitory action. This mutual photoreversibility
led to the prediction that there were two forms of photochromic pigment, and that the peaks corresponded to
the shifting absorption peaks of the two forms of the
pigment.
With mutual photoreversibility available as an assay
criterion, and with the aid of an instrument produced by
Karl Norris for the detection of small absorbancy
changes in optically dense material such as apple
fruits10, it was possible to demonstrate the physical existence of the pigment in vivo and in vitro, and ultimately to isolate a highly purified blue-green protein,
appropriately named phytochrome11. We now know that
there is a family of phytochromes consisting of at least
five members differing in cellular location, absorption
properties and physiological action. Collectively, they
control an impressive array of developmental processes,
including seed germination, seedling deetiolation, formation of chlorophylls, anthocyanins and other pigments, circadian rhythms and flowering.
A detailed account of subsequent progress in elucidating the purification and isolation of phytochrome, the
nature and structure of its protein and chromophore
moieties, its cellular locations, its possible modes of
action and physiological significance is described in a
remarkable book by a non-scientist, Linda Sage, entitled
146
Pigment of the Imagination12. Sage, a writer and resident of St. Louis, was asked by Joseph Varner, then a
professor at Washington University in St. Louis, to undertake the writing of the history of phytochrome. With
support from the USDA, she travelled around Europe,
Japan and the United States for over two years, interviewing and interpreting the contributions of most of
the researchers in the field of phytochrome. Included in
her narrative are contributions from several prominent
individuals whom I had the privilege of introducing to
the field of ‘phytochromology’ while serving as a professor in the Department of Biology at Yale University.
These included my former graduate student Masaki Furuya, my former post-doctoral associates Harry Smith,
William S. Hillman, Ruth L. Satter and Bruce A. Bonner, and my DuPont (where I was a consultant) colleagues Franklin E. Mumford and Edward L. Jenner.
Recent work and interpretations on the mechanism of
action of phytochromes are reviewed in a subsequent
article of this special section by Sharma.
Flavoprotein photoreceptors: The blue-light receptors,
by contrast, have been isolated only during the last two
or three years, partly because some early conclusions
about their nature were faulty. In the 1930s, it was
widely believed that carotenoids were the receptors for
phototropism, on the basis of a similarity between their
absorption spectra and the action spectrum for phototropism13. Because carotenoids function in animal vision, and because of a general belief in the relatedness
of functions of similar biochemical compounds, this
conclusion was firmly established14. For example, Erwin
Bünning, who had become justly famous for his discoveries in the field of circadian rhythmicity, published a
series of papers entitled ‘Phototropismus und Carotinoide’ over a 20-year period starting from l937 (refs 15–
18). He concluded, on the basis of considerable circumstantial evidence, including action spectra, that the receptor pigments for phototropism and associated blue-lightmediated plant photo-reactions were carotenoids.
In 1949, just fifty years ago, I challenged Bünning’s
conclusions when I discovered that photo-activated riboflavin could oxidize the phytohormone IAA in vitro19, and proposed that this reaction might explain the
phototropic curvature in grass coleoptiles known to be
associated with auxin asymmetry. I showed further that
action spectra in the blue (made with the Beltsville
monochromator under the supervision of Sterling
Hendricks) could fit flavoproteins as well as the carotenoids, and reasoned that it would be difficult, on the
basis of action spectra in the visible alone, to discriminate between the two putative photoreceptors20. The
subsequent discovery of an action peak near 370 nm
favoured the flavin type of photoreceptor. When later
experiments by Briggs et al.21 showed that flavinmediated photo-destruction of IAA could not explain
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phototropism, since the amount of auxin in responding
organs remained constant, many people rejected the
flavin photoreceptor theory entirely. But I had also
pointed out that certain amino acids, like tryptophan
and histidine, as well as polypeptides and enzymes containing them, as well as bacteriophages, were also substrates for photo-activated flavins20. Thus, flavins might
function as photoreceptors even if auxin destruction was
not involved in phototropic curvature.
The idea of physiologically significant photoreception
by flavins remained unproved until very recently, when
the lengthy and elegant experiments of Briggs and coworkers with a nonphototropic mutant of Arabidopsis
led to isolation of ‘phototropin’, a flavoprotein which is
now accepted as the photoreceptor for this process22. In
the same way, a flavoprotein photoreceptor has recently
been identified by Cashmore et al.23 as responsible for
the blue-light-mediated inhibition of stem growth and
for DNA lyase activity in Arabidopsis. Both workers
referred to my early work suggesting flavin photoreception. Pterins have also been invoked as photoreceptors24
in other blue-light-sensitive systems; since riboflavin
may be considered as a benzpteridine, this idea is consonant with the flavin hypothesis. The subject of bluelight-mediated reactions is reviewed in an essay by
Khurana in this special section.
I have often been asked why I virtually abandoned research on flavins as photoreceptors almost 50 years ago,
when the idea had generated such interest and seemed
so attractive. I can only reply that at the time of this
discovery, I was a 29-year old untenured Research Fellow, and that those expressing strong opposition to my
theory included George Wald (later a Nobelist for work
on carotenoid photochemistry during vision), Kenneth
Thimann (the eminent auxin researcher), Frits Went (the
discoverer of auxin), Folke Skoog (later the discoverer
of kinetin) and Erwin Bünning (the distinguished exponent of circadian rhythms). When, after submitting a
grant renewal application to the National Science Foundation, I was warned that in view of the strong criticism
my work was receiving from respected authorities in the
field, future work on this topic would probably have
difficulty obtaining funding, I found it expedient to
switch to a different line of research. As it turned out,
this was a serious error. Young researchers of today
should probably take note of this object lesson.
Plant hormones
The cultivation of single cells and of most plant tissues
requires greater knowledge of plant growth factors than
was generally available before the early 1950s. Went’s
auxin2 had been characterized as 3-indoleacetic acid
(IAA) by Kögl et al25, and shown to be required for the
growth and division in culture of a wide variety of plant
cells not derived from an organized meristem. (In such
CURRENT SCIENCE, VOL. 80, NO. 2, 25 JANUARY 2001
structures, apical regions synthesize their own auxin,
making exogenous additions superfluous.) Groups under
both F. C. Steward at Cornell and F. K. Skoog at Wisconsin sought the identity of additional growth factors
from natural sources such as coconut milk, which had
been found by Johannes van Overbeek et al.26 to favour
the growth of immature Datura embryos that would
otherwise not grow in culture. Investigating other natural sources of growth promoters such as yeast extract
and herring sperm, the Wisconsin group isolated from
the latter an unnatural substance named kinetin27, an
artifact formed during the autoclaving of isolated DNA.
In the presence of auxin, kinetin promoted the division
of cells of tobacco pith. This finding led to a search for
naturally-occurring analogues of kinetin, resulting ultimately in the isolation by Letham and others28 of several ‘cytokinins’, in coconut milk and elsewhere. The
molar ratios of auxin and cytokinin in tissue cultures of
tobacco pith and other plant materials determines
whether the products of cell division remain as undifferentiated callus or whether they differentiate into rootor bud-primordia29. Cytokinins also turn out to promote
protein synthesis in excised leaves, and thus to mimic
the effect of roots attached to otherwise isolated leaves.
Gibberellin
Japanese investigators had long focused on a growth
factor produced when the fungus Gibberella fujikuroi
invaded rice to produce the elongated stems characteristic of the bakana-e disease30. Eventually, gibberellic
acid (GA) was isolated from higher plants as well as
fungal media31, shown to be physiologically active, and
to be one of a large number of metabolically interrelated
‘gibberellins’ whose biosynthetic pathway is now reasonably well understood32. The roles of gibberellins in
the control of germination33 and dwarfism34 were later
discovered and investigated, and their role in flowering
and other physiological phenomena is now under active
investigation.
Abscisic acid
Abscisic acid (ABA) was named and discovered in the
context of leaf abscission by Addicott and coworkers35
at Davis, California. At virtually the same moment in
history, the same compound was isolated by Wareing
and colleagues36 at Aberystwyth, Wales, who were
studying bud dormancy. In the hindsight of history,
ABA turns out to be much more closely associated with
seed and bud dormancy than with leaf abscission, and
its suggested designation as ‘dormin’ or dormic acid
seems more appropriate. Most recently, ABA has been
shown to be involved in response to water stress, and to
function in the gain and loss of solutes during changes
in stomatal guard cell turgor37.
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Ethylene
It had long been appreciated that unsaturated hydrocarbon gases used as fuels in the heating of greenhouses
could cause dramatic changes in growth and morphology, exemplified by leaf abscission and by the ‘triple
response’ of etiolated pea seedlings, i.e. shortening and
fattening of epicotyls, ageotropic behaviour and formation of the apical hook38. Later, the role of ethylene in
hastening the ripening processes of fruits was found to
be associated with its natural occurrence in plants and
its rapid rise in titer following a sudden ‘climacteric’
rise in respiratory release of carbon dioxide39. Eventually, its formation from methionine through the intermediacy of ACC (1-aminocyclopropane 1-carboxylic
acid) was described40. The mechanism of ethylene action in a variety of physiological responses, from tropisms to leaf fall, is currently under investigation.
In the last decade, considerable attention has been
paid to several other substances with presumed hormonal characteristics. Jasmonic acid, synthesized from
linolenic acid in the membrane by a lipoxygenaseinitiated chain of reactions, activates genes controlling
the synthesis of proteinase inhibitors involved in defence reactions41. A peptide named systemin is said to
play a similar role42, possibly triggering the biosynthesis of jasmonic acid. Salicylic acid also occurs naturally
in plant tissues and plays a role in the high respiratory
rate, leading to the warming of the fleshy spathe of
skunk-cabbage and related plants43. It may also be related to resistance to plant pathogens and to various
stress reactions44. Polyamines such as putrescine and
spermidine are ubiquitous in plants, and are considered
by some to control, or at least to be importantly involved in such processes as mitosis, response to stress
and antagonism of ethylene-induced fruit ripening45.
Diminution of polyamine titer through inhibitor-induced
blockage of polyamine biosynthesis leads to inhibition
of the above processes. Polyamines are discussed further below.
One conspicuous failure in phytohormone research is
our continuing inability to define the nature of the
flower-inducing stimulus, named florigen, more than
three decades ago46. The physiological evidence for the
existence of such a substance is overwhelming: photoperiodic induction of one region of an appropriate plant
leads to flowering in the non-induced region as well;
the same effect can be produced by grafting an induced
region to an uninduced region. A single leaf is effective
as a donor when it is photo-induced and grafted to a
defoliated receptor; translocation of the stimulus is apparently through the phloem, and the rate of movement
resembles that of sucrose47. While we know of substances, including phytohormones like gibberellin and
ethylene, whose application can lead to flowering in
some uninduced plants, their limited action compels us
148
to reject them as candidates for florigen. Since both
long-day and short-day plants are capable of inducing
the other type to flower by grafting, we must assume
that the floral stimulus is the same or at least functionally equivalent in both types. To date, no candidate
compound fulfills all these requirements, although ethylene and gibberellin are effective in some plants. Thus,
despite much recent molecular genetic information
about the control of flowering in Arabidopsis and other
plants48, we remain in the dark about the nature of the
transmissible floral inducer that triggers the flowering
process. The presently dominant theory is that flowering is controlled not by a single substance, but by the
interactions of a multiplicity of unconnected stimuli,
some endogenous and some environmental. By contrast,
a simple theory, based on interactions among three
genes, satisfactorily explains the formation of sepals,
petals, stamens and carpels49.
A discussion of the mechanism of action of plant
hormones can be found in a subsequent article in this
special section by Johri and Mitra.
Plant organ tissue and cell culture
In the early l930s, P. R. White50 had learned how to
cultivate excised root tips on media containing yeast
extract; shortly thereafter, F. C. Robbins and J. Bonner
separately repeated this finding for tomato and pea
roots, and defined the effective components of the yeast
extract as thiamin (needed by all roots), pyridoxine
(needed by tomato roots) and niacin (needed by pea
roots). At about the same time Gautheret and Nobécourt
in France had experienced success in achieving the potentially unlimited and largely undifferentiated growth of
slices of carrot root. The product here was largely undifferentiated callus containing isolated whorls of tracheids.
Two decades later, F. C. Steward would demonstrate that
even a single isolated cell of this material could be made
to regenerate embryoids and even the entire organism,
thus proving that each cell contains the entire functional
genome of the plant, and is potentially totipotent. Developments in this field are well summarized and referenced
in the monograph edited by Street51.
After another decade or so, in which many other investigators succeeded in regenerating entire plants from
single cells (for details, see ref. 52), the ultimate step in
plant regeneration was successfully made. It was
shown, especially in the Solanaceae53, that even isolated
protoplasts, enzymatically deprived of their cellulosic
walls and stabilized by osmotica, could give rise to the
entire plant. Such naked protoplasts became useful in a
great variety of experiments, ranging from purely
physiological to developmental and genetic. It was even
found possible to fuse protoplasts of different origins by
chemical or electrical means to achieve ‘parasexual
plant hybridization’54.
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Investigations on crown gall disease led to the discovery of the crucial roles of Agrobacterium tumefaciens and of wounding in the tumefaction process55.
Grafting demonstrated the transmissibility of the ‘tumour-inducing principle’ and culture of excised secondary crown gall tumours revealed auxin autotrophy and
an absence of recoverable bacteria56. Based on these
observations and comparisons between virulent and
avirulent strains of the bacterium, it was not long until a
plasmid was isolated that carried genes for tumour initiation as well as for synthesis of hormones and other
substances57. This was one of the first documented
cases of interspecific gene transfer, and Agrobacterium
became one of the first effective tools for producing
transgenic organisms.
Circadian rhythms
Many creatures change their appearance and morphology in response to light, and we have already described
the importance of light–dark cycles on floral initiation.
Frequently correlated with these effects on flowering
are rapid, reversible change in leaf attitude. For example, some plants have leaves that droop in darkness and
are erect in the light, but if maintained in continuous
darkness after exposure to a diurnal light–dark cycle,
the leaves continue to rise and fall in a rhythmic fashion58. Erwin Bünning noted that the effect of a light
period on leaf movement, flowering and other phenomena depended not only on its intensity, duration and
wavelength, but also on the time of day or night the
light is administered. When an artificially lengthened
dark period was probed by a light flash given at various
times, the response curve described a sine wave, with
alternating periods of promotion and inhibition, the
peaks occurring approximately 24 h apart59. Such phenomena, involving endogenous, self-sustained oscillations were called circadian rhythms; and such rhythms
have since been described for animals60,61, plants62 and
micro-organisms63. The period of such rhythms is independent of temperature (implying the operation of a
compensatory mechanism), but is markedly affected by
light absorbed by either a phytochrome or a flavoprotein receptor. It is now believed that photoperiod works
by ‘resetting the clock’ that drives the rhythmic behaviour, thus keeping the organism in synchrony with the
external environment. Several genes showing rhythmic
behaviour in transcription have now been isolated64.
In support of an apparently lost cause –
polyamines
Over two decades ago, Ravindar Kaur-Sawhney and I
were investigating the culture of cereal protoplasts,
when we made an observation that led us and others to a
CURRENT SCIENCE, VOL. 80, NO. 2, 25 JANUARY 2001
long series of investigations on polyamines (summarized in ref. 65). In order to obtain protoplasts from
young oat leaves, we had to peel off the epidermis before exposing the leaf to cellulase, which liberated the
mesophyll cells as protoplasts into the surrounding medium containing hypertonic mannitol. We noted that
leaves pre-exposed to arginine, or to any of the polyamines derived from arginine, greatly stabilized the
integrity and green colour of the peeled leaves and the
protoplasts derived from them66. We thus proposed that
polyamines might be involved in the control of leaf senescence. Later investigations by one of my graduate
students, Hector Flores, showed that protoplasts contained much higher titers of putrescine than the leaf
cells from which they were derived67. This was found
due to de novo synthesis of arginine decarboxylase
(ADC), one of the two enzyme systems involved in the
formation of putrescine from arginine, the other being
ornithine decarboxylase, ODC. We later found that numerous other stresses, such as low pH68, potassium limitation69 and heavy metals70 produced the same effect
through the same mechanism, and investigations by
others added to the list of stresses that activated putrescine accumulation in stressed cells. By the mid l980s,
high putrescine titer had come to be recognized as a
criterion of stress in cereals and some other plants as
well. Yet, to our surprise and chagrin, most subsequent
conferences and reviews on the physiology of stress in
plants have not mentioned polyamines at all! The cause
of this omission remains a mystery to me, a mystery
which I leave to future workers to resolve.
Polyamines seem also to be involved in the control of
cell division and morphogenesis, being required for embryoid formation from cultured carrot cells71, for flower
production in ‘thin-layer’-derived cultures of tobacco
callus72, for the growth73 and prevention of senescence
and ethylene production of tomato fruits74, for export of
the florigenic stimulus from induced leaves of the longday plant Sinapis alba75 and for bolting and flowering
in Arabidopsis thaliana76. A summary of the literature
on polyamines as related to reproductive activities and
stress in plants has recently appeared77, and a masterful
overall summary of polyamine biochemistry and physiology in all kinds of creatures has been written by Seymour Cohen78, a pioneer in this field.
Since polyamines and enzymes regulating their biosynthesis and metabolism are present in all cells, some
basic function of these compounds is implied. Their
titer in cells is usually in the fractional millimolar
range, and they must be applied as multiple millimolar
solutions to elicit physiological reactions. This is, of
course, at least a thousand-fold higher concentration
than the effective range for traditional plant hormones,
and may be one reason why the possible physiological
regulatory role of these compounds is so seldom considered. At physiological pH, they are strongly cationic,
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SPECIAL SECTION: PLANT MOLECULAR BIOLOGY
and can couple with many cellular polyanions such as
DNA and RNA, as well as certain proteins and surface
components of membranes. In doing so, they change the
configuration of the anionic molecule with which they
couple, often with important consequences. This includes activation of kinases and phosphatases79 and the
conversion of active B-configuration DNA chains to the
inactive Z-form80. Polyamines may also be covalently
linked to proteins through the action of transglutaminase81, again with possible consequences for functional
alteration. When presented to plant cells, polyamines
tend to accumulate in vacuoles, inhibiting the uptake of
K+ (ref. 82). In vitro, they are powerful inhibitors of
proteases83. Because of these properties and demonstrated physiological actions, it is likely that polyamines
will turn out to be important in some physiological
regulations. If this prediction is correct, many textbooks
and reviews will have to be altered.
A look into the future
Whither plant physiology in the next millennium, especially in areas dealing with growth and development?
Realizing that prediction is an inexact art, I will be conservative here, while expecting progress in three directions: one toward the molecular and cellular analysis of
growth and development, a second toward agricultural
and ecological considerations, and a third toward ethical concerns. The molecular–cellular movement will
bring us closer to explanations of basic problems in
plant physiology, while the second and third will lead
us to confront urgent societal problems for which we
have relevant information and expertise, and for which
equitable and ethical solutions are required.
One obvious trend is the increasing use of molecular
genetic ideas and techniques to obtain information on
mechanisms controlling the attainment of morphogenetic patterns. It has already been brilliantly successful
in arriving at a rational and provable series of rules for
development of each of the four whorls of flower
parts84. The imminent completion of the Arabidopsis
genome project will make possible the identification,
cloning and transgenic use of new regulatory genes.
Comparative genomics will also make it possible to
specify the function of some genes and their protein
products. The use of individual cells and protoplasts in
transgenic experiments in which regeneration of organs
or entire plants must be attained will also obviously
increase, providing a parallel boost in research on cell
and tissue culture.
While the search for new regulatory factors in plants
has probably almost run its course, recent discoveries
about jasmonic acid and salicylic acid strongly indicate
that some new ones still remain to be discovered. One
class with probable significance will be the peptide
150
hormones, like systemin42. In general, investigations
into the reasons for specific obligate relations between
parasites or saprophytes and their host plants should
give new leads.
More attention needs to be paid to the physiology of
roots and the rhizosphere. For example, while the importance of mycorrhizae is partially understood, their
possibilities for further improving plant growth have
not been well explored.
As the world’s human population grows beyond 6 billion toward still uncharted heights, agricultural productivity must obviously also increase if these extra people
are to be fed, since there is virtually no new land to
bring into production. In the past 50 years, waves of
improvement in productivity have come from conventional genetics, fertilizers, irrigation, plant protection
and the recent ‘green revolution’. Yet now, we see the
necessity for a new wave, since we are facing a new
‘ceiling’ probably imposed by existing genomes. Will
the new increase come from transgenic plants, from
improved agronomic technology, from improved understanding of plant–plant interaction, or some other direction?
Along with improved productivity, we must strive for
better nutritional quality, an endeavour in which gene
transfer techniques will almost certainly prove important. With the introduction of transgenes into agricultural crops, we must concern ourselves with problems
voiced, especially in Europe, about their possible harmful ecological side-effects. Will such genes escape to
the wild, creating ‘superweeds’? Will introduced genes
be allergenic or toxic to human recipients or to the ecosystem? Should transgenically modified crops be so
labelled for the consumers who are concerned? These
problems, relating to the health of individuals and entire
ecosystems, must be attacked and solved if geneticallymodified foods are to become important. Other environmental problems, like those resulting from the massive use of nitrogenous fertilizers and the increasing
salinization of irrigated soils, must also be addressed.
One ethically distressing result of plant physiological
research in the last century was the massive use of defoliants as weapons of war in south-east Asia from about
1967 to 1971 (ref. 85). About 45 million litres of a formulated mixture containing herbicidal levels of 2,4-D
and 2,4,5-T, designated in military parlance as Agent
Orange, were sprayed by United States airplanes onto
about 1.7 million hectares of mainly upland forests and
mangroves and some crop lands in Vietnam. The object
was to reveal enemy combatants hidden by the otherwise dense vegetation, but among the unwanted sideeffects were a series of ecologically damaging events,
including a loss of minerals from the ecosystem by increased nutrient dumping, soil erosion and gulleying,
replacement of valuable species like teak by useless and
noxious weeds like scrub bamboo, and the destruction
CURRENT SCIENCE, VOL. 80, NO. 2, 25 JANUARY 2001
SPECIAL SECTION: PLANT MOLECULAR BIOLOGY
of some food crops by deliberate or accidental drifting
of the aerial spray. In addition, it was later discovered
that Agent Orange contained potentially toxic levels of
dioxins, amongst the most noxious materials ever synthesized86. Vietnam veterans, whose service led to their
being sprayed, received many millions of dollars in
compensation from the manufacturers of Agent Orange
after a lengthy court battle87, but no recompense has yet
been offered to Vietnamese mothers who may have produced teratogenic babies or suffered from other toxic
effects.
Since the use of these chemical weapons, the largest
such use in military history, may have violated important international treaties88, and in any event led to numerous public protests at home and abroad, their use in
future warfare appears unlikely. Their use has led to a
proposal that ecocide be designated as a war crime to
parallel the crime of genocide, justly condemned at the
Nuremberg trials following the Nazi era. It would seem
appropriate for plant physiological associations to register their opposition to any such antisocial use of materials originally designed to enhance agricultural
productivity and practice.
The growing importance of industrial research laboratories has brought new dimensions into plant physiology, including new jobs, new money for academic
research, and new practices, such as the use of patents
and the concomitant restriction in the flow of information. Decisions about what research to pursue and how
much to publish are now frequently made not by scientists, but by corporate officials relatively untrained in
science and perhaps more interested in increased profitability than in increased knowledge. Our scientific organizations must take note of this trend and propose
methods to deal with it.
Finally, as evidence for global climatic change becomes more and more convincing, plant physiologists
need to concern themselves with its possible agricultural consequences. Should we develop crops with
higher temperature optima for growth and productivity?
Or simply look for new crops to fit the new climatic
situation? Should the emphasis be on C3 or C4 plants?
Clearly, plant biology faces many important problems
and opportunities ahead. We need to continue to train
able scientists to discharge these responsibilities in an
efficient, ethical and timely manner.
1. Garner, W. W. and Allard, H. A., J. Agric. Res., 1920, 18, 553–
607.
2. Went, F. W., Recl. Trav. Bot. Néerl., 1928, 25, 1–116.
3. Miller, E. C., Plant Physiology, McGraw Hill, New York,
1931.
4. Taiz, L. and Zeiger, E., Plant Physiology, Sinauer, Sunderland,
Massachusetts, 1998, 2nd edn.
5. Hamner, K. C. and Bonner, J., Bot. Gaz., 1938, 100, 388–431.
6. Hendricks, S. B., Annu. Rev. Plant Physiol., 1970, 21, 1–
10.
CURRENT SCIENCE, VOL. 80, NO. 2, 25 JANUARY 2001
7. Parker, M. W. et al., Bot. Gaz., 1946, 108, 1–26.
8. Parker, M. W., Hendricks, S. B. and Borthwick, H. A., Bot.
Gaz., 1950, 111, 242–252.
9. Flint, L. H. and McAlister, E. D., Smithson. Misc. Collect., l937,
96, 1–8.
10. Butler, W. L. and Norris, K. H., Arch. Biochem. Biophys., l960,
87, 31–40.
11. Siegelman, H. W. and Firer, E. M., Biochemistry, 1964, 3, 418–423.
12. Sage, L. C., Pigment of the Imagination, Academic Publ, New
York, 1992.
13. Johnston, E. S., Smithson. Misc. Collect., 1934, 92, 1–17.
14. Wald, G., Am. Sci., 1954, 42, 88–95.
15. Bünning, E., Planta, 1937, 26, 719–736.
16. Bünning, E., Planta, 1937, 27, 148–158.
17. Bünning, E., Planta, l937, 27, 583–610.
18. Bünning, E., Z. Bot., 1955, 43, 167–174.
19. Galston, A. W., Proc. Natl. Acad. Sci. USA, 1949, 35, l0–17.
20. Galston, A. W., Science, 1950, 111, 619–624.
21. Briggs, W. R., Tocher, R. D. and Wilson, J. F., Science, 1957,
126, 210–212.
22. Christie, J. M. et al., Proc. Natl. Acad. Sci. USA, l999, 96,
8779–8783.
23. Cashmore, A. R. et al., Science, 1999, 284, 760–765.
24. Senger, H. (ed.), Blue Light Effects in Biological Systems,
Springer Verlag, Berlin, 1984.
25. Kögl, F., Chem. Weekbl., 1932, 29, 317–318.
26. van Overbeek, J., Conklin, M. E. and Blakeslee, A. F., Science,
l941, 94, 350–351.
27. Miller, C. O., Annu. Rev. Plant. Physiol., 1961, 12, 395–408.
28. Letham, D. S., Annu. Rev. Plant. Physiol., 1967, 18, 349–364.
29. Skoog, F. K. and Miller, C. O., Symp. Soc. Exp. Biol., 1957, 11,
118–131.
30. Stowe, B. B. and Yamaki, T., Annu. Rev. Plant Physiol., 1957,
8, 181–216.
31. Brian, P. W., Int. Rev. Cytol., 1966, l9, 229–266.
32. Macmillan, J., Encycl. Plant Physiol. n.s. 9:, Springer Verlag,
Berlin, 1980.
33. Varner, J. E. and Chandra, G. R., Proc. Natl. Acad. Sci. USA,
1964, 52, 100–106.
34. Phinney, B. O., Biol. Plant., 1985, 27, 172–179.
35. Addicott, F. T. and Lyon, J. L., Annu. Rev. Plant. Physiol.,
1969, 20, 139–164.
36. Wareing, P. F. and Saunders, P. F., Annu. Rev. Plant Physiol.,
1971, 22, 261–288.
37. Allan, A. C. et al., Plant Cell, 6, 1319–1328.
38. Pratt, H. K. and Goeschl, J. D., Annu. Rev. Plant Physiol., l969,
20, 541–584.
39. Burg, S. P. and Burg, E. A., Bot. Gaz., 1965, 126, 200–204.
40. Yang, S. F., in Biochemistry and Physiology of Plant Growth
Hormones (eds Wightman, F. and Setterfield, G), Runge Press,
Ottawa, l969.
41. Creelman, R. A. and Mullet, J. E., Annu. Rev. Plant. Physiol.
Plant. Mol. Biol., l997, 48, 355–381.
42. Pearce, G. et al., Science, 1991, 253, 895–898.
43. Raskin, I., Turner, I. M. and Melander, W. R., Proc. Natl. Acad.
Sci. USA, 1989, 86, 2214–2218.
44. Shulaev, V., Silverman, P. and Raskin, I., Nature, 1997, 385,
718–721.
45. Slocum, R. D., Kaur-Sawhney, R. and Galston, A. W., Arch.
Biochem. Biophys., 1984, 235, 283–303.
46. Chailakhian, M. Kh., Bot. Rev., 1975, 41, 1–29.
47. Bernier, G., Annu. Rev. Plant Physiol. Plant Mol. Biol., 1988,
39, 175–219.
48. Meyerowitz, E. M. and Somerville, C. (eds), Arabidopsis, Cold
Spring Harbor Laboratory Press, Plainview, New York, 1994.
49. Weigel, D. and Meyerowitz, E. M., Cell, 1994, 78, 203–209.
50. White, P. R., Plant Physiol., 1934, 9, 585–600.
151
SPECIAL SECTION: PLANT MOLECULAR BIOLOGY
51. Street, H. E. (ed.), Plant Tissue and Cell Culture, University of
California Press, Berkeley, 1973.
52. Evans, D. A. et al. (eds), Handbook of Plant Cell Culture,
Macmillan, New York, 1983, vols 1 and 2.
53. Cocking, E. C., Nature, 1960, 187, 962–963.
54. Carlson, P. S., Smith, H. H. and Dearing, R. D., Proc. Natl.
Acad. Sci. USA, 1972, 69, 2292–2294.
55. Braun, A. C., Annu. Rev. Plant Physiol., 1962, 13, 533–558.
56. Nester, E. et al., Annu. Rev. Plant Physiol., 1984, 35, 387–413.
57. Chilton, M. D. et al., Cell, l977, 11, 263–271.
58. Sweeney, B. M., Rhythmic Phenomena in Plants, Academic
Press, New York, 1969.
59. Bünning, E., The Physiological Clock, Springer-Verlag, Berlin,
1973.
60. Pittendrigh, C. S., Cold Spring Harbor Symp. Quant. Biol., l960,
25, 73–86.
61. Brown, F. A. and Webb, H. M., Physiol. Zool., 1948, 21, 371–
381.
62. Satter, R. L. and Galston, A. W., Annu. Rev. Plant Physiol.,
1980, 32, 83–110.
63. Hastings, J. W. and Sweeney, B. M., Proc. Natl. Acad. Sci.
USA, l957, 43, 804–811.
64. Millar, A. J. and Kay, S. A., Cell, 1991, 3, 541–550.
65. Tiburcio, A. F., Kaur-Sawhney, R. and Galston, A. W., in The
Biochemistry of Plants (eds Stumpf, P. K. and Conn, E. E.),
1990, vol. 16, 283–325.
66. Kaur-Sawhney, R. and Galston, A. W., Plant Cell Environ.,
1979, 2, 189–196.
67. Flores, H. E. and Galston, A. W., Science, 1982, 217, 1259–
1261.
68. Young, N. D. and Galston, A. W., Plant Physiol., l983, 71, 767–
771.
69. Young, N. D. and Galston, A. W., Plant Physiol., 1984, 74,
331–335.
152
70. Weinstein, L. H. et al., Plant Physiol., 1986, 82, 641–645.
71. Feirer, R. P., Mignon, G. and Litvay, J. D., Science, 1984, 223,
1433–1435.
72. Tiburcio, A. F., Kaur-Sawhney, R. and Galston, A. W., Plant
Cell Physiol., 1988, 29, 1241–1249.
73. Mizrahi, Y. and Heimer, Y., Plant Physiol., 1982, 54, 367–
368.
74. Dibble, A. R. G., Davies, P. J. and Mutschler, M. A., Plant
Physiol., 1988, 86, 338–340.
75. Havelange, A. et al., Physiol. Plant., 1996, 96, 59–65.
76. Applewhite, P. B., Kaur-Sawhney, R. and Galston, A. W.,
Physiol. Plant., 2000.
77. Galston, A. W. et al., Bot. Acta, l997, 110, 197–207.
78. Cohen, S. S., A Guide to the Polyamines, Oxford University
Press, Oxford, 1998.
79. Liang, T. et al., Biochim. Biophys. Acta, 1978, 542, 430–441.
80. Wang, A. H. et al., Nature, 1979, 282, 680–686.
81. Clarke, D. D. et al., Arch. Biochem. Biophys., l959, 79, 338–
354.
82. De Agazio, M., Giardina, M. C. and Grego, S., Plant Physiol.,
l988, 87, 176–178.
83. Balestreri, E. et al., Arch. Biochem. Biophys., 1987, 552, 460–
463.
84. Coen, E. S. and Meyerowitz, E. M., Nature, 1991, 353, 31–
37.
85. Westing, A. H. (ed.) Herbicides in War: The Long-Time Ecological and Human Consequences, Taylor and Francis, London,
1984.
86. Blair, E. H. (ed.), Chlorodioxins: Origin and Fate, Advances in
Chemistry Series 120, American Chemical Society, Washington
DC, 1973.
87. Schuck, P. H., Agent Orange on Trial, Belknap-Harvard University Press, Cambridge, MA, 1986.
88. Galston, A. W., Ann. NY Acad. Sci., 1972, 196, 223–235.
CURRENT SCIENCE, VOL. 80, NO. 2, 25 JANUARY 2001